Physiological, biochemical and molecular changes due … · Physiological, biochemical and...

Physiological, biochemical and molecular changes due to seed priming in Cucumber

(Cucumis sativus L.)

1

Dr. SOWMYA, K.J University of Agricultural Sciences,

National Seed Project(Crops) GKVK, Bangalore, India.

Email: [email protected] 6/26/2013

2

Introduction Seed priming is a pre-sowing treatment that involves controlled hydration of seeds, sufficient to allow pre-germinative metabolic events to take place and to restrict radicle protrusion through the seed coat (Heydecker et al., 1973).

Ø Common name: Cucumber

Ø Scientific name: Cucumis sativus

Ø Family : Cucurbitaceae

Ø Origin: India

Ø Ploidy level: 2n = 14

Ø Mode of pollination : Cross pollinated

Ø Period of crop growth : Annual

Origin and Taxonomy

3 6/26/2013 UAS, Bangalore, India

☻To know the physiological, biochemical and molecular changes due to seed invigoration

☻To know the protein marker (s) associated with

invigoration

4

Objectives

6/26/2013 UAS, Bangalore, India


Material and Methods • Vigour levels (V): V1- Fresh seeds - high vigour (>90%) V2- low vigour (<60%)

• Treatments (T): T1- Control T2- Water T3- Ethrel @ 100 ppm T4- KNO3 @ 1% T5- PEG 6000, -1.5 MPa T6- Cow dung slurry (1:1 w/v) T7- PSB • Total treatment combinations: 2 x 7 = 14 • Replications: Three • Design: Factorial CRD

Treatment details

Ø Seed moisture content (%) [ISTA (2007)] Ø First count germination (%) [ISTA (2007)] Ø Final count germination (%) [ISTA (2007)] Ø Mean germination time (in days) [Ellis and Roberts (1981)] Ø Time to 50 % germination (T50) [ Coolbear et al. (1990). ] Ø Speed of germination (BRI) [(Bartlett, 1973)] Ø Germination energy (%) [Wang (2008) ] Ø Coefficient of velocity of germination (%) [Nichols and Heydecker (1968)] Ø Mean seedling length (cm) Ø Mean seedling dry weight (mg) Ø Seedling vigour index -I Ø Seedling vigour index -II [Abdul-Baki and Anderson (1973) ]


Observations : Physiological changes

ØElectrical conductivity (dSm-1) [ISTA (2007)]

ØTotal soluble seed protein (%) [Lowry et al. (1951)]

ØTotal soluble sugars (%) [Dubois et al. (1951)]

ØTotal dehydrogenase activity [Perl et al. (1978)]

ØAmylase activity [Bernfeld (1955) ]

ØPeroxidase activity [Sadasivam and Manickam (2008)]

ØCatalase activity [Goth (1991)]

ØDNA content (µg/g of seed) [Doyle and Doyle (1990) ] 7 6/26/2013 UAS, Bangalore, India

Observations : Biochemical changes

Ø Electrophoretic characterization of total soluble seed protein through NATIVE- PAGE

Ø Electrophoretic characterization of total soluble seed protein through SDS- PAGE [Laemmeli (l970)]

Ø Elecrophoretic analysis of esterase (EST) and [Glaszman et al. (1988) ]

Ø Elecrophoretic analysis of Peroxidase isozyme (POX) [Glaszman et al. (1988) ]


Molecular changes

6/26/2013 UAS, Bangalore, India 9

Experimental

Results Physiological

Biochemical & Molecular aspects

Treatments First count germination (%) Final count germination (%)

HV LV Mean HV LV Mean

T1 71.33 49.00 60.17 89.33 57.33 73.33

T2 94.00 69.33 81.67 95.33 71.67 83.50

T3 95.33 81.33 88.33 95.33 82.67 89.00

T4 97.33 86.00 91.67 98.00 86.67 92.33

T5 87.33 67.33 77.33 93.00 71.00 82.00

T6 90.67 77.33 84.00 92.67 82.00 87.33

T7 93.33 80.00 86.67 95.33 82.00 88.67

Mean 89.90 72.90 81.40 94.14 76.19 85.17

SEm± CD

(P=0.01) CV (%) SEm±

CD (P=0.01)

CV (%)

V 0.696 2.71 0.498 1.94 T 1.302 5.08 3.91 0.932 3.64 2.68 V x T 1.841 7.19 1.318 5.15

Table 1. First and final count germination as influenced by vigour levels and priming treatments in cucumber

Treatments (T): T1- Control; T2- Water ; T3- Ethrel @ 100 ppm; T4- KNO3 @ 1%; T5- PEG 6000, -1.5 MPa; T6- Cow dung slurry (1:1 w/v); T7- PSB


Treatments Mean germination time(days) CV of germination (%) SVI

HV LV Mean HV LV Mean HV LV Mean

T1 2.38 3.23 2.81 42.02 30.99 36.50 689 351 520

T2 1.26 1.94 1.60 79.92 52.81 66.36 981 710 845

T3 1.36 1.69 1.53 73.95 60.26 67.10 1086 895 991

T4 1.24 1.54 1.39 81.07 66.00 73.54 1182 980 1081

T5 1.49 2.72 2.11 69.47 37.01 53.24 996 661 829

T6 1.39 1.93 1.66 72.07 51.71 61.89 985 880 933

T7 1.37 1.81 1.59 74.41 55.54 64.97 1063 885 974

Mean 1.50 2.12 1.81 70.41 50.62 60.51 998 766 882

SEm± CD

(P=0.01) CV (%) SEm±

CD (P=0.01)

CV (%) SEm± CD

(P=0.01) CV (%)

V 0.04 0.18 1.75 6.86 8.7 34.2

T 0.08 0.33 1.74 3.28 12.84 3.30 16.3 64.0 4.55

V x T 0.12 0.48 4.64 NS 23.1 90.5

Table 2. Mean germination time, coefficient of velocity of germination and Seedling Vigour Index (SVI) based on seedling dry weight as influenced by vigour levels and priming treatments in cucumber



control (dry) water

Ethrel @ 100ppm KNO3 @1%

PEG @-1.5 MPa

Cowdung slurry

PSB

Influence of various priming treatments on germination in cucumber

6/26/2013 12 UAS, Bangalore, India

Control Water PEG KNO3 Ethrel Cowdung Slurry PSB

Treatments Conductivity of seed leachate (dS/m) Total dehydrogenase activity (A480)

HV LV Mean HV LV Mean

T1 22.54 43.37 32.96 2.215 1.292 1.753

T2 18.63 21.67 20.15 2.547 2.001 2.274

T3 15.87 23.86 19.87 2.894 2.006 2.450

T4 14.80 22.63 18.72 2.995 2.117 2.556

T5 15.59 20.75 18.17 2.854 1.995 2.424

T6 19.55 32.66 26.11 2.125 1.885 2.005

T7 18.35 31.56 24.96 2.114 1.734 1.924

Mean 17.91 28.07 22.99 2.535 1.861 2.198

SEm± CD

(P=0.01) CV (%) SEm± CD

(P=0.01) CV (%)

V 0.043 0.17 0.005 0.019

T 0.081 0.32 0.86 0.009 0.036 1.02

V x T 0.115 0.45 0.013 0.051

Table 3. Conductivity of seed leachate, total dehydrogenase activity, as influenced by vigour levels and priming treatments in cucumber



Treatments Peroxidase activity

(∆A 436/min/g of seed) Catalase activity

(mMol H2O2 degraded/min) DNA content

(µg/g of seed)

HV LV Mean HV LV Mean HV LV Mean

T1 0.128 0.136 0.132 36.80 32.00 34.40 34.25 30.61 32.43

T2 0.322 0.444 0.383 46.15 49.20 47.68 35.57 32.66 34.11

T3 0.492 0.537 0.514 59.80 58.90 59.35 35.90 33.39 34.64

T4 0.573 0.665 0.619 69.30 65.00 67.15 36.20 33.94 35.07

T5 0.481 0.506 0.493 53.80 51.50 52.65 35.36 32.95 34.15

T6 0.426 0.496 0.461 46.85 43.90 45.38 34.89 32.57 33.73

T7 0.221 0.268 0.244 39.60 39.75 39.68 34.90 33.64 34.27

Mean 0.377 0.436 0.406 50.33 48.61 49.47 35.29 32.82 34.06

SEm± CD

(P=0.01) CV (%) SEm±

CD (P=0.01)

CV (%) SEm± CD

(P=0.01) CV (%)

V 0.002 0.010 0.170 0.66 0.049 0.19

T 0.005 0.018 2.81 0.318 1.24 1.57 0.092 0.36 0.66

V x T 0.007 0.026 0.450 1.76 0.130 0.51

Table 4. Peroxidase activity, catalase activity and DNA content as influenced by vigour levels and priming treatments in cucumber



Vigour Levels: V1- Fresh seeds - high vigour (>90%); V2- low vigour (<60%); Treatments: T1- Control; T2- Water;nT3- Ethrel @ 100ppm; T4- KNO3 @ 1%; T5- PEG 6000, -1.5 Mpa; T6- Cow dung slurry and T7- PSB

Influence of seed priming on native protein profile in cucumber

26 Polymorphic bands (Rm value: 0.033 to 0.846).

V1T1 V1T2 V1T3 V1T4 V1T5 V1T6 V1T7 V2T1 V2T2 V2T3 V2T4 V2T5 V2T6 V2T7

0.000

0.285

0.357

0.428

0.500

0.571

0.642

0.714

0.785

0.857

0.928

1.000

0.071

0.142

0.214

Zymogram of native protein due to seed priming in cucumber


3kDa

6.5kDa

14.3kDa

20kDa

29kDa

43kDa

68kDa

97.4kDa

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Influence of seed priming on total soluble seed protein (SDS-PAGE) profile in cucumber

M V1T1 V1T2 V1T3 V1T4 V1T5 V1T6 V1T7 V2T1 V2T2 V2T3 V2T4 V2T5 V2T6 V2T7


27 bands (Rm value: 0.100 to 0.966) polymorphic regions : B, C, E, F, G and H.

0.083

0.500

0.583

0.666

0.750

0.833

0.916

1.000

0.000

0.166

0.250

0.333

0.416

Region A

Region B

Region C

Region D

Region E

Region F

Region G

Zymogram of SDS-PAGE protein due to seed priming in cumber


M V1T1 V1T2 V1T3 V1T4 V1T5 V1T6 V1T7 V2T1 V2T2 V2T3 V2T4 V2T5 V2T6 V2T7


Influence of seed priming on Esterase (EST) isozyme in cucumber


Polymorphic bands with Rm values: 6 (0.242), 7 (0.257), 8 (0.300), 16 (0.635), 17 (0.678), 19 (0.721), 20 (0.771), 21 (0.785), 22 (0.800), 23 (0.864) and 24 (0.892)

Zymogram of esterase (EST) isozyme due to seed priming in cucumber

0.000

0.285

0.357

0.428

0.500

0.571

0.642

0.714

0.785

0.857

0.928

1.000

0.071

0.142

0.214




Influence of seed priming on Peroxidase (POX) isozyme in cucumber



Monomorphic bands (Total of six bands having Rm value of 0.062 to 0.565)

0.000

0.285

0.357

0.428

0.500

0.571

0.642

0.714

0.785

0.857

0.928

1.000

0.071

0.142

0.214

Zymogram of peroxidase (POX) isozyme due to seed priming in cucumber



The first count & CV of germination

• Significantly higher in HV seeds compared to LV seeds

KNO3 primed seeds

• Higher first count & CV of germination compared to unprimed seeds in both HV & LV seeds

HV seeds had higher TDH activity, TSP and DNA content compared to LV seeds Activities also higher in KNO3 primed seeds in contrast with unprimed seeds

CONCLUSION


TSS of seed leachate

• Significantly lower in HV seeds than LV seeds • Lower in KNO3 primed seeds compared to unprimed seeds

The amylase, catalase & peroxidase activities

• Significantly higher in KNO3 primed seeds compared to unprimed • HV seeds showed increased activity of these enzymes compared to LV

Conversely, TSP, esterase & peroxidise enzyme profiles revealed polymorphism with respect to expression of peptides due to priming induced proteins.

CONCLUSION Continued.

The enhanced biochemical activities, particularly in low vigour seeds might have resulted in the enhanced seed quality attributes following priming in cucumber.

6/26/2013 UAS, Bangalore, India 25 25

acknowledgement

Ø DST, Ministry of Science and technology, New Delhi, India Ø Department of Seed Science and Technology, UAS, Bangalore, India Ø Indian Institute of Horticultural Research, Hessaraghatta, Bangalore, India

THANK YOU for your attention

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